Borrelia burgdorferi, the cauative agent of Lyme disease, is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi contains abundant linear and circular plasmids and there is growing evidence that these plasmids carry genes critical for adaptation. A major obstacle to studying the functions of specific plasmids and gene products in the infectious cycle of B. burgdorferi has been an inability to genetically manipulate pathogenic strains. To address this issue, we have developed conditions for site-directed mutagenesis in B31-MI, the infectious B. burgdorferi strain whose genome was recently sequenced. We inactivated several plasmid and chromosomal genes in B31-MI and determined that clones carrying these mutations were not infectious for mice. However, we found extensive heterogeneity among clones and mutants derived from B31-MI based on colony phenotype, growth rate, plasmid content and protein composition. This high degree of clonal polymorphism within B31-MI complicates the assessment of mutants and indicates that it is not an appropriate strain background for genetic studies of infectious B. burgdorferi. In order to establish a well-defined isogenic clone for targeted mutagenesis studies, we derived a wild type clone from B31-MI that was infectious for mice and transformable. Our studies also demonstrate that the instability of the B. burgdorferi genome mandates careful monitoring of plasmid content of derived mutants and complementation of inactivated genes in order to perform genetic studies with this pathogen. We have inactivated several plasmid-encoded genes in an infectious clone and demonstrated that these clones and the complemented controls have retained all plasmids. Thus we have finally succeeded in genetically manipulating a pathogenic B. burgdorferi clone. This allows comparisons of mutant and isogenic wild type clones in order to identify the functions of specific gene products. In collaboration with a consortium of extramural investigators, we have developed an array of all B. burgdorferi genes and are using this array to compare global patterns of gene expression under different growth conditions and between mutant and wild type clones. We have established an experimental infectious cycle in which we are assessing the competence of mutant clones lacking specific genes or plasmids for infectivity in both the tick vector and mammalian host, and for transmission between them. We are further characterizing the functions of specific B. burgdorferi gene products through biochemical analyses of isogenic mutant and wild type clones.